Actin-binding domain of mouse plectin
Crystal structure and binding to vimentin
Jozef S
ˇ
evc
ˇ
ı
´
k
1
, L’ubica Urba
´
nikova
´
1
,Ju
´
lius Kos
ˇ
t’an
1,2
, Lubomı
´
r Janda
2
and Gerhard Wiche
2
1
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic;
2
Institute of Biochemistry and

filament networks.
Plectin is a versatile cytoskeletal linker protein of very
large size that is abundantly expressed in a wide variety
of mammalian tissues and cell types. As a cytolinker par
excellence it has been found in association with various
cytoskeletal structures and it has been shown to interact with
a variety of distinct proteins on the molecular level (reviewed
in [1]). Plectin’s putative role as a mechanical stabilizing
element of cells was fully confirmed when EBS-MD, a severe
skin blistering disease combined with muscular dystrophy,
was traced to defects in the plectin gene [2], with plectin-
deficient mice showing a similar phenotype [3].
Similar to other members of an emerging family of
structurally related cytolinker proteins, referred to as the
plakins [4,5], plectin displays a multidomain structure
composed of a central  200 nm long rod segment flanked
by globular domains [1]. The interaction sites of various
cytoskeletal proteins have been mapped to opposite ends of
the molecule optimizing its potential as a cytoskeletal linker
protein. One of the better characterized interaction domains
of plectin is its N-terminal actin-binding domain (ABD)
which is of the canonical type, comprising two tandemly
arranged calponin homology (CH) domains, CH1 and
CH2. This relatively small domain ( 30 kDa) is found
in many actin-binding and cytolinker proteins, such as
a-actinin, dystonin, fimbrin, spectrin/fodrin, dystrophin and
utrophin, to name a few.
However, in certain aspects the ABD of plectin seems to
be unique. Analysis of the plectin gene from mouse revealed
the unusual high number of 14 alternatively spliced first

4-phenyl-2-butanone.
Note: The atomic coordinates and structure factors (code 1SH5 and
1SH6) have been deposited in the Protein Data Bank, Reasearch
Collaboratory for Structural Bioinformatics, Rutgers University,
New Brunswick, NJ ( />(Received 21 December 2003, revised 26 February 2004,
accepted 18 March 2004)
Eur. J. Biochem. 271, 1873–1884 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04095.x
dystrophin and utrophin with F-actin through direct
binding to CH domains, although the physiological rele-
vance of this is not clear [11,12]. Furthermore, CH domains
contain specific binding sites for phosphoinositides and PIP
2
has been shown to modulate the actin-binding activity of
a-actinin [13] and plectin [14]. Another intriguing feature,
so far characterized only for plectin ABD, is the direct
interaction with the cytoplasmic tail domain of the integrin
b4 subunit [15,16].
For a number of proteins with either a single CH
subdomain or tandemly arranged CH subdomains (ABD),
interactions with intermediate filament (IF) proteins have
been reported. Calponin has been shown to interact with
desmin, the major IF protein of smooth and striated muscle
[17–19], and for fimbrin a colocalization with vimentin was
observed in cultured macrophages [20]. In both cases it was
suggested that interactions were mediated by CH domains.
Based on this, the idea arose that the interaction of
CH domains with IF subunit proteins may represent a
highly conserved function common to CH protein family
members.
As of recently, crystal structures of ABDs have been

(pGR147); ple1aABD starting with exon 1a-encoded
sequences, but without exon 2a sequences; ple6–9 cDNA
(pGR103) starting at the last codon of exon 5 and extending
to half of exon 9 (ATC CGG); and ple4–8 (pDS19) cDNA
starting with the first in-frame ATG in exon 4 and extending
close to the end of exon 8 (GCA CAG). The differences
were that ple4–8 cDNA was subcloned into pBN120
through EcoRI and NdeI sites and that ple1aABD cDNA
was subcloned into expression vector pGR66, a modified
derivate of pBN120 missing a His-tag.
A fragment corresponding to repeat 5 of mouse plectin
(1039 bp) was generated by PCR (forward primer,
5¢-GGAATTCCGCGGTCTCCGCAAGC-3¢; reverse primer
5¢-GGAATTCAAGCGTACCAGCGCGGTAC-3¢), using
mouse plectin cDNA (rat accession number X59601) as a
template. This fragment was subcloned into expression
vector pBN120 resulting in plasmid pKAB1.
Plasmid pFS129 encoding full length mouse vimentin
(accession number M26251) without tag has been described
previously [27]. Plasmids pFS2 and pFS3, encoding the
N-terminus of vimentin (Met1–Glu94) and the rod domain
(Phe95–Ile411) of vimentin, respectively, were prepared
similar to pKAB1, using primer pairs pFS2 forward
(5¢-CCGGAATTCATGTGGACCAGGTCTGTG-3¢), and
pFS2 reverse (5¢-CCGGAATTCCTCAGTGTTGATGG
CGTC-3¢), and pFS3 forward (5¢-CCGGAATTCTTCAA
GAACACCCGC-3¢), and pFS3 reverse (5’-CCGGAATT
CAATCCTGCTCTCCTC-3’), and mouse vimentin cDNA
as a template. Plasmid pGP1 encoding the rod and
C-terminus of vimentin (Phe95–Glu466) was obtained by

ptoethanol, 0.4 m
M
phenylmethanesulfonyl fluoride (solu-
tion A) and after centrifugation (40 000 g,15min,4°C)
supernatants were directly applied to a 10 mL DEAE
Sepharose CL-6B column equilibrated with solution A.
Bound protein was eluted with a 60 mL gradient of NaCl
(0–0.3
M
) in solution A, and aliquots of fractions (2 mL)
were analyzed by SDS/PAGE. Vimentin-containing frac-
tions were pooled, diluted 1 : 5 (v/v) with 50 m
M
sodium
formate, pH 4.0, 8
M
urea, 10 m
M
2-mercaptoethanol,
0.4 m
M
phenylmethanesulfonyl fluoride (solution B) and
applied to a 10 mL CM Sepharose CL-6B column in
solution B. After washing, bound vimentin was eluted with
a linear gradient of KCl (0–0.3
M
) in solution B, and
aliquots were stored at )20 °C. His-tagged, truncated
versions of vimentin and plectin ABD were purified by
affinity chromatography as described [15].

vimentin or ple1cABD/2a (both at concentrations of
5 lgÆmL
)1
)in4.3m
M
Na
2
HPO
4
,1.4m
M
KH
2
PO
4
,
137 m
M
NaCl, 2.7 m
M
KCl, 1 m
M
EGTA, 2 m
M
MgCl
2
,
0.1% (v/v) Tween 20, 1 m
M
dithiothreitol, pH 7.5. After 1 h

100 lgÆmL
)1
). The digest was then immediately loaded
onto a pleABD/2a Sepharose column equilibrated with
10 m
M
Tris/HCl, pH 7.5, 0.5 m
M
MgCl
2
,0.2m
M
dithio-
threitol, 25 m
M
NaCl, 50 lgÆmL
)1
TPCK (solution E).
Bound protein was eluted with a linear gradient of
25–400 m
M
NaCl in solution E.
Crystallization
Two crystalline forms (I and II) were prepared by the
hanging-drop vapor diffusion method. Monoclinic crystals
(form I), belonging to the P2
1
space group with two
molecules in the asymmetric unit, were grown from 4 lL
drops containing equivalent amounts of protein and

crystals the procedure was repeated using solutions with
lower concentrations of protein (10 mgÆmL
)1
)andprecip-
itant [8% (w/v) PEG 8000]. The precipitant solution was
enriched with dioxane (2%, v/v) to reduce twinning
tendency of crystals. The first microcrystals were used as
seeds. Crystals reached dimensions of up to 0.8 mm after
1–2 days.
Data collection and processing
The collection and processing of X-ray data from crystal
form I (structure I) were described previously [28]. Data
from crystal form II (structure II) were collected to 2.0 A
˚
resolution at 100 K on EMBL X-11 beamline at the
DORIS storage ring, DESY, Hamburg. Crystals were
soaked stepwise in cryoprotectant prepared from precipi-
tant solution enriched with glucose (6, 12, 18 and 24%, w/v)
before flash-freezing. Conditions for data collection were
optimized using the program
BEST
[32]. Data collection
statistics are summarized in Table 1 (data from crystal form
I are included for comparison). Dimensions of the unit cell,
crystal symmetry and molecular mass of the protein gave a
crystal packing density V
M
of 2.5 A
˚
3

1
2
1
2
1
Unit cell parameters
a(A
˚
) 55.31 32.52
b(A
˚
) 108.92 51.23
c(A
˚
) 63.75 144.72
b (°) 115.25 90
Mosaicity 0.3 0.4
Solvent content (%) 60 50
Completeness (%) 96.2 (71.7) 97.6 (86.3)
R(I)
merge
a
(%) 6.0 (40.0) 3.8 (31.0)
I/r(I) 22.2 (2.1) 33.9 (3.5)
a
R(I)
merge
¼ S/I )<I>/SI, where I is an individual intensity
measurement and <I> is the average intensity for this reflection
with summation over all data.

ing the model, the program
ARP
/
WARP
in the mode
WARPNTRACE
[37] was used. The program automatically
built a model consisting of 378 out of 490 residues (molecules
A and B) with a connectivity index of 0.92. The remaining
residues, including those forming the loop connecting the
CH1 and CH2 subdomains, were built manually using the
program
O
[38] running on a Silicon Graphics Station.
Structure I was refined with the program
REFMAC
5.
Sparse matrix was used as the method of minimization.
Refinement of the structure was altered with correcting
the amino acid sequence and building the parts which
were different from those of utrophin and which were not
built by
WARPNTRACE
. The structure was refined against
95% of the data, the remaining 5% (randomly excluded
from the full data set) were used for crossvalidation in
which R
free
was calculated to follow the progress of
refinement [39]. After each refinement cycle,

pleABD/2a, is unique, as it contains a five amino acid-long
sequence (Fig. 1) inserted by differential splicing of a short
exon (exon 2a) between the first two exons of the ABD [6].
Comprising 245 residues, the analyzed recombinant protein
contained pleABD/2a as a 237 residues-long fragment
(amino acids 181–417; EMBL accession number AF
188008) flanked by six amino-terminal (GSHMEF) and
two carboxy-terminal (EF) residues (added as cloning
requirement). The amino acid residues in the structures
are numbered from 1 to 245 according to the sequence of
the recombinant protein, i.e. amino acids with numbers
7–243 in the structure correspond to 181–417 in the AF
188008 sequence.
Two crystal structures of pleABD/2a were determined.
One of them, structure I was derived from a monoclinic
crystal containing two protein molecules (A and B) in the
asymmetric unit, the other, structure II, from an orthorhom-
bic crystal, containing one molecule in the asymmetric unit
(for details see Materials and methods). As found in structure
I, molecule A, pleABD/2a is an a-protein consisting of 11
helices: a1 (residues 8–25), a2 (48–58), a3 (70–86), a4 (96–
100), a5 (104–118), a6 (134–145), a7 (165–172), a8 (181–186),
a9 (189–204), a10 (212–215), and a11 (222–235) (Figs 1 and
2A). Helices a1–a5 form the CH1 and helices a6–a11 the
CH2 subdomain. The subdomains are connected by a
flexible 15 residues-long loop (119–133). For five N-terminal
(GSHME) and eight C-terminal residues (RVPGAQEF) of
both molecules in structure I there was no electron density
observed. In structure II there was no electron density for the
first seven (N-terminal) nor for the last eight (C-terminal)

B molecule 39.3 –
side-chain A molecule 46.6 39.8
B molecule 44.2 –
Waters 46.2 38.9
Rms deviation from ideal geometry (target values are given in
parentheses)
Bond distances (A
˚
) 0.03 (0.02) 0.04 (0.02)
Bond angles (°) 2.02 (1.94) 2.59 (1.94)
Chiral centers (A
˚
3
) 0.22 (0.20) 0.21 (0.20)
Planar groups (A
˚
) 0.01 (0.02) 0.02 (0.02)
Main-chain bond B-values (A
˚
2
) 2.16 (1.50) 2.38 (1.50)
Main-chain angle B-values (A
˚
2
) 3.44 (2.00) 3.57 (2.00)
Side-chain bond B-values (A
˚
2
) 4.70 (3.00) 5.27 (3.00)
Side-chain angle B-values (A

, corres-
ponding to  3% (380 A
˚
2
)ofthesurfaceofeachisolated
molecule (11 800 A
˚
2
). This was far below the minimum of
9% required for classification of a dimer as a protein complex
[42], suggesting that the crystallographic dimer hardly could
exist in solution. Moreover, structure II has confirmed that
pleABD/2a can exist as a monomer in solution.
The contacts between molecules in the crystal lattice
apparently did not change the orientation of the CH1 with
respect to the CH2 subdomain in spite of the fact that the
loop connecting the two subdomains theoretically could
allow various orientations including those found in
utrophin. It can be concluded that the conformation of
pleABD/2a as found in structures I and II is stable and not
subject to conformational changes due to different crystal
packing.
Quality of protein structure models
The final R and R
free
factors for structure I were 15.3 and
19.4% (Table 2). There were two protein molecules (A and
B) and 188 solvent molecules in the asymmetric unit. The
Ramachandran plot [43] calculated by the program
PRO-

factors were 20.2
and 29.9% (Table 2). The structure contained one protein
Fig. 2. Ribbon representation of the pleABD/
2a structure I and comparison with utrophin and
fimbrin ABDs. (A) Stereo view of pleABD/2a.
Individual helices are numbered. (B) Crystal-
lographic dimer as seen in the asymmetric
unit. The views are related by 90° rotation
around a horizontal axis. Molecules A and B
are shown in red and blue, respectively.
(C) Overlap (stereo view) of CH1 and CH2
subdomains of pleABD/2a molecule A with
the CH1 subdomain of utrophin molecule A
and the CH2 subdomain of utrophin molecule
B. Utrophin molecules are colored in light
(molecule A) and dark blue (molecule B). The
plectin molecule is in red. (D) Overlap (stereo
view)ofpleABD/2a (red) with fimbrin ABD
(green). Figures were generated using the
program
MOLSCRIPT
[52].
1878 J. S
ˇ
evc
ˇ
ı
´
k et al.(Eur. J. Biochem. 271) Ó FEBS 2004
and 58 solvent molecules in the asymmetric unit and in the

for which distances between corresponding Ca atoms
exceeded 1.75 A
˚
(Fig. 3B, dashed line), the rms displace-
ment values were 0.25 (IB/IA, 232 Ca atoms), 0.48 (II/IA,
225 atoms), and 0.62 A
˚
(HP/IA, 224 atoms). The largest
differences in Ca positions (up to 2.5 A
˚
) were found in the
surface loop region, which was not unexpected considering
that each molecule has a different environment in the
crystal.
Least squares superpositions of mouse plectin with the
corresponding subdomains of human plectin, utrophin,
dystrophin and fimbrin are summarized in Table 3. In these
superpositions only the CH1 (8–119) and CH2 (134–236)
subdomains from structure I molecule A were used; the
connecting segment was excluded as its conformation differs
substantially among the various proteins compared (helix
in utrophin and dystrophin, helix–loop in fimbrin, and loop
in plectin). Furthermore, as the ABDs of utrophin and
dystrophin adopt an open conformation (contrary to the
plectin ABD), the CH1 domain from utrophin molecule A
and the CH2 domain from molecule B were used in the
overlap. The different numbers of Ca atoms involved in
Fig. 3. Comparisons of mouse and human
ABDs. Average main chain B factor values of
mouse ABDs (A), and differences between Ca

from utrophin (dystrophin) molecule A, CH2 from utrophin
(dystrophin) molecule B (see Figs 2C,D, and relevant text).
Ó FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur. J. Biochem. 271) 1879
superpositions (reflecting the degree of structural similarit-
ies) clearly showed highest similarity of mouse plectin with
human plectin and lowest with fimbrin (Table 3). The
nearly identical conformations of the pleABD/2a structure
and the corresponding subdomains of the A and B
molecules of utrophin and dystrophin probably have not
arisen by chance and may have significance for functional
properties of these ABDs.
The ABD of plectin binds to vimentin
The IF protein vimentin has been reported to specifically
interact with the CH1 subdomain of the first (N-terminal) of
fimbrin’s two ABDs [20]. In light of the extensive structural
resemblance of fimbrin and plectin ABDs it was therefore of
interest to assess IF-binding activity of the plectin ABD.
Moreover, a second IF protein interaction domain at the N
terminus in addition to its C-terminal IF-binding site [26]
would raise plectin’s functional versatility, particularly as a
cytoskeletal crosslinking element. To assess the plectin
ABD–vimentin interaction, in vitro overlay assays were
performed. Ple1aABD, an ABD version of plectin preceded
by a sequence encoded by exon 1a[6], pleABD/2a and
truncated versions of the plectin ABD missing half of the
CH1 domain (ple4–8), or the complete CH1 domain (ple6–
9) were immobilized on nitrocellulose membranes and
overlaid with full-length vimentin. In agreement with
previously reported findings [20] all proteins, except the
one missing the entire CH1 domain (ple6–9) and the

pleABD/2a with filamentous vimentin in sedimentation
assays (data not shown) it seems that the plectin ABD
interacts with vimentin in the same way.
Fig. 4. Overlay of various plectin ABD versions with full-length vimen-
tin. Recombinant versions of the plectin ABD starting with exon 1a-
encoded sequences (ple1aABD), or starting with exon 2-encoded
sequences and containing 2a-encoded sequences (pleADB/2a), or
lacking part of (ple4–8), or the whole CH1 domain (ple6–9), as well as
a fragment corresponding to the repeat 5 domain of plectin (positive
control), and BSA (negative control) were subjected, in duplicate, to
12.5% SDS/PAGE. Proteins on one gel were blotted onto a nitrocel-
lulose membrane and overlaid with recombinant full-length mouse
vimentin (B), proteins on a second gel were stained with Coomassie
Blue (A). All proteins, except for ple6–9 and BSA, showed significant
binding to vimentin.
1880 J. S
ˇ
evc
ˇ
ı
´
k et al.(Eur. J. Biochem. 271) Ó FEBS 2004
To confirm the specificity of the plectin ABD–vimentin
interaction and to more precisely map vimentin’s plectin
ABD-binding site, vimentin purified in urea was kept in its
soluble (tetrameric) form by dialysis into solution D (see
Material and methods). The protein was than subjected to
limited chymotryptic digestion and fragments generated
were applied to a pleABD/2a-Sepharose affinity column.
Elution and SDS/PAGE of bound proteins revealed that a

pleABD/2a are structurally similar to the fimbrin ABD,
although these domains share only little sequence identity.
The fimbrin and plectin ABDs have a similar closed
conformation, differing from the open conformation des-
cribed for the ABDs of dystrophin and utrophin [21–24]. It
is interesting to note that until now, sequences correspond-
ing to exons 2a and 3a of mouse plectin have been identified
neither in other family members, nor in human plectin. In
this view, insertion of exon 2a into the sequence of
human pleABD/2a as reported [24] seems without rational
explanation.
Although the ABDs of fimbrin, utrophin and a-actinin
are related, they appear to have different effects on F-actin
conformation upon binding and thus may use different
mechanisms of association [25,45,46]. Up to now a consen-
Fig. 6. Affinity-binding of proteolytically derived fragments of vimentin to pleABD/2a. Partial chymotryptic digestion of vimentin and affinity-
chromatography of fragments on a pleABD2a-Sepharose column was carried out as described in the text. SDS/PAGE of eluted fractions is shown.
Lanes 1–11, wash fractions; 12–27, salt-gradient elution of bound proteins. Co, sample loaded onto column. The molecular mass of size markers run
in left-most lane is indicated. The18 kDa fragment of vimentin binding to pleABD/2a mapped to the N-terminal part of the vimentin rod domain,
as determined by MALDI-TOF mass spectrometry.
Fig. 5. Overlay of recombinant vimentin fragments with plectin ABD.
Recombinant versions of vimentin subdomains were immobilized on
nitrocellulose membranes as described in Fig. 4, and overlaid with the
ple1cABD/2a. Vim, full-length vimentin; VimN, N-terminal domain;
VimR, rod domain; VimRC, vimentin without N-terminal domain;
VimNR, vimentin without C-terminal domain. To detect vimentin-
bound plectin ABD, plectin isoform 1c-specific antibodies [31] were
used. Note the strong binding of plectin ABD to full-length vimentin
and VimNR, but only weak or no binding to other vimentin fragments.
Ó FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur. J. Biochem. 271) 1881

The ABDs of dystrophin and utrophin crystallized as
antiparallel dimers [21,22], whereas fimbrin and human
plectin ABDs crystallized in monomeric form [23,24]. When
pleABD/2a was crystallized at pH 9, we also found two
molecules in the asymmetric unit [28]. However, conditions
at pH 6.5 led to the formation of crystals with only one
pleABD/2a molecule in the asymmetric unit. The assess-
ment of the contact area between molecules A and B in
pleABD/2a crystals obtained at pH 9 (crystal form I)
showed that these molecules do not interact strongly enough
to form dimers which could exist also in solution. Therefore,
the crystallographic dimer observed in the asymmetric unit
was probably an artefact of crystallization rather than a
dimerization product.
Although CH subdomains forming a canonical ABD
structurally seem to be highly conserved, data are accumu-
lating that show functional diversity of CH1 and CH2
subdomains [8]. It was reported that proteins containing
either single CH subdomains (calponin), or more complex
ABDs (fimbrin) can interact with IF proteins. Calponin
binds to desmin, the major IF protein in smooth and
skeletal muscle [17–19] and fimbrin interacts with and
colocalizes with vimentin in filopodia, retraction fibres,
and podosomes at the ventral surface of cultured macro-
phages [20]. In both cases there is evidence that binding
occurred to IF subunit proteins in their nonfilamentous
state [17–20]. Fimbrin was unable to bind to polymerized
vimentin in cosedimentation assays and binding occurred at
a stoichiometry of 1 : 4, suggesting that the IF protein was
in its tetrameric form [20].

NR used in [20] overlapped by a few amino acid residues.
With this consideration, the results of our overlay assay
were consistent with the findings reported in [20]. Using as
an alternative method affinity-binding of proteolytic frag-
ments of vimentin in combination with mass spectrometry,
we found that plectin bound to a  18 kDa fragment
corresponding to ÔCoil 1Õ [49], the N-terminal part of the a-
helical rod domain of vimentin. This is in agreement with
similar experiments in which the calponin-binding site was
restricted to the N-terminal part of the desmin rod domain
[19]. However, in the overlay assay, the rod domain of
vimentin (VimR) alone showed little binding to plectin. As
fragments obtained by partial proteolytic digestion of
properly folded full-length vimentin are more likely to
preserve the structure of the native protein than recombi-
nantly prepared fragments, we assume the true plectin
ABD-binding site of vimentin to be localized in the N-
terminal part of its rod domain. Binding to the evolutionary
highly conserved CH domains could be a common feature
of IF proteins in general. Likewise, considering that the a-
helical coiled-coil structure of vimentin’s rod domain is
highly conserved in all IF protein family members, plectin’s
ABD may bind also to other IF proteins, such as desmin.
The functional significance of the plectin ABD–vimentin
interaction remains elusive. The primary activity of the
ABD supposedly is actin-binding, a function demonstrated
for both fimbrin and plectin [14,50]. The CH domain of
calponin, on the other hand, doesn’t seem to be required
for actin-binding as calponin interacts with actin via its
C-terminal domain [51]. Thus, with an IF protein-binding

¨
nther Rezniczek, Daniel Spazierer and Ferdinand
Steinbo
¨
ck, for providing various reagents and for valuable discussions.
We are grateful to the EMBL Hamburg team for providing us with
synchrotron facilities and for help in data collection. We would also like
to thank the European Community for supporting J.S. and L.U.
through the Access to Research Infrastructure Action of the Improving
Human Potential Programme to the EMBL Hamburg Outstation
(contract HPRI-CT-1999–00017). This work was supported by the
Slovak Academy of Sciences Grant 2/1018/21 (J.S. and L.U.), Austrian
Science Research Fund Grant P14520 (G.W.), and an Austrian Federal
Ministry of Education, Science, and Culture Research Contract (G.W.).
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